Identification Of Cell Wall Degrading Enzymes Of Geotrichumcitri-aurantii And Its Role In Pathogenesis

Citrus is an important economic tree species in south China.It is the main way for many fruit growers to increase their income.It plays an important role in the economic development of agriculture in south China.Citrus sour rot is one of the main and difficult diseases to control after harvest.In recent years,it has a trend of outbreak year by year in various citrus producing areas.The main reason for the lack of commercial chemical fungicides and unstable effect of citrus sour rot is that the pathogenesis of Geotrichum citri-aurantii is not fully understood.At present,the research on the pathogenic mechanism of G.citri-aurantii is mostly in the introduction of the pathogenic pathway,the pathogenesis of the disease and the development of the course of the disease.There is no systematic research on the potential pathogenic factors.Some studies have shown that the cell wall degrading enzyme is an important pathogenic factor of postharvest pathogenic fungi of fruits and vegetables,but there are few reports on the cell wall degrading enzyme of G.citriaurantii.In this paper,the genome of the mycelium of G.citri-aurantii was sequenced and several potential genes of cell wall degrading enzyme were annotated.The kinds of cell wall degrading enzyme secreted by the pathogen in 4 days were determined by LC-MS/MS.The infection process of G.citri-aurantii on citrus fruits was observed by scanning electron microscopy(SEM),and the expression of cell wall degrading enzyme genes was determined by real-time PCR.The activity of cell wall degrading enzymes and the changes of cell wall polysaccharides(cellulose,pectin and hemicellulose)in citrus fruits during pathogen infection were determined.The main results of this paper are as follows:(1)The genome of G.citri-aurantii pathogen was sequenced,about 27 MB of sequences were assembled,GC content was 38.62 %,and 5,708 coding genes were predicted.Six cell wall degrading enzyme genes were identified from the genome of G.citri-aurantii,including polygalacturonase gene,?-1,4-glucanase gene,?-1,6-mannanase gene,?-1,3-glucanase gene,?-1,3-glucosidase gene,and ?-1,2-mannosidase gene.It was identified by LC-MS/MS that polygalacturonase,?-1,3-glucanase and ?-1,2-mannosidase can be secreted by the pathogen on the 4th day.(2)The SEM results showed that after inoculation of citrus fruit for 4 h(4 hpi),the spores of G.citri-aurantii expanded,the buds germinated in 6 h,and the buds began to destroy the orange peel tissue in 8 h.With the extension of inoculation time,the spores germinated into mycelia.After 8 hpi,the mycelia broke and more conidia were produced,the pathogen invaded from the fruit wound,and the invasion of mycelium to the host was obviously observed at 12 hpi.(3)The results of quantitative PCR showed that the expression of polygalacturonase gene at 6 h was 4.76 times that of the control,and then the expression was on the rise,with the highest expression at 48 h,53.70 times that of the control.The expression level of ?-1,4-glucanase gene at 6 h was 1.14 times that of the control,the expression level of ?-1,4-glucanase gene at 24 h was increased and 12.02 times that of the control,followed by a slight decrease.The expression levels of ?-1,6-mannanase and ?-1,2-mannosidase genes were low,There was no expression of ?-1,3-glucosidase gene and ?-1,3-glucanase gene.(4)The activities of polygalacturonase,?-1,4-glucanase,?-1,3-glucanase and ?-1,6-mannanase increased first and then decreased during the infection of citrus fruit by G.citri-aurantii,which was significantly higher than that of the control.Among them,the highest activity of ?-1,3-glucanase and ?-1,6-mannanase was 20.95±0.12 U/mg and 11.16±0.85 U/mg at 24 h,the highest activity of ?-1,4-glucanase was 36.28±0.48 U/mg at 48 h,and the highest activity of polygalacturonase was 21.95±0.99 U/mg at 72 h,while the activity of ?-1,2-mannosidase and ?-1,3-glucosidase was close to that of control.(5)After inoculation with G.citri-aurantii for 12 h,the citrus fruit showed obvious disease,and the diameter of the disease spot increased the fastest in 48-96 h.The results of polysaccharide content determination showed that the infection of G.citri-aurantii damaged the cell wall structure of citrus peel,and caused the degradation of cellulose,total pectin and hemicellulose to different degrees,and the degradation of total pectin was greater than that of cellulose than that of hemicellulose.The degradation rate and pathogenesis are related to the role of cell wall degradation enzymes.Among them,polygalacturonase plays an important role in the degradation of pectin,?-1,4-glucanase and ?-1,3-glucanase play an important role in the degradation of cellulose,while ?-1,6-mannanase plays an important role in the degradation of hemicellulose.Above all,this study preliminarily defined the types,action time sequence of secretory cell wall degrading enzymes and their effects on cell wall polysaccharides of citrus fruits during the infection of G.citri-aurantii,which indicated that cell wall degrading enzymes played an important role in the pathogenesis of citrus sour rot.The results of this study will provide theoretical basis and reference for the prevention and control of citrus postharvest sour rot.